This application relates to a method and apparatus for rapid gel electrophoresis and fluorescence detection of a complex mixture of fluorophore labeled proteins or nucleic acids.
Polyacrylamide gel electrophoresis (PAGE) separation of organic molecules is now routinely performed. Current Protocols in Molecular Biology, Chap. 10, John Wiley & Sons (1994). A polyacrylamide gel provides a suitably insoluble sieve so as to permit the separation of organic molecules in solution by size and conformation as they are drawn through the sieve under electromotive force. Such separation of organic molecules provides valuable insights into their structures and functions. For example, PAGE separation can separate two polypeptides of the same size but of different isoforms or polypeptides only 100 daltons different in size (Current Protocols, 1994). Another use for PAGE is in separation of nucleic acids based on size of fragments, such as in the extremely important application of DNA sequence determination. Maniatis, Molecular Cloning, A Laboratory Manual, 2nd ed., 1987.
Early methods for detection of separated products on an electrophoresis gel were carried out after the separation was completed. To the extent these techniques were automated in an apparatus, the apparatus generally including a mechanism for moving the gel in stages relative to a detector. For example, U.S. Pat. No. 4,343,991 discloses a sample detection apparatus in which stained bands within a gel are detected by transporting the gel between an array of optical fibers supplying incident light and an array of optical fibers collecting transmitted light. Information was collected in steps along the whole length of the gel. Devices of this type have the advantage that long sample collection times can be used when necessary to ensure detection of low intensity bands in the gel. However, they also have several drawbacks. In particular, because the gel is analyzed only after separation is completed, the degree of separation is necessarily a compromise between the desire to observe fast migrating bands (which run off the end of the gel if the electrophoresis proceeds too long) and the desire to separate slow moving band (which are still grouped together if the electrophoresis run is too short). In addition, the use of distinct separation and detection processes significantly lengthened the time required to complete an analysis. Thus, the art has generally sought the ability to detect bands on an electrophoresis gel during the separation.
When the electrophoresed molecules are labeled with a detectable signal, it is possible to detect the separations of molecules in real time. Since the first description of a real-time nucleic acid separation method and apparatus by Smith et al., Sequence Detection in Automated DNA Sequence Analysis", Nature 321:674-679 (1986), the technology for so-called automated DNA sequencing has expanded rapidly. Several automated DNA sequencing apparatuses are commercially available. Methods and apparatus for sequencing of DNA are described in U.S. Pat. Nos. 4,811,218; 4,881,812; 5,062,942; 5,091,652; 5,108,179; 5,122,345; 5,162,654; 5,171,534; 5,190,632; 5,207,880; 5,213,673; 5,230,781; 5,242,567; 5,290,419; 5,294,323; 5,307,148; 5,314,602; 5,324,401; and 5,360,523 which are incorporated herein by reference.
Unfortunately, the existing apparatuses are inadequate for use of PAGE for emerging clinical diagnostic purposes such as diagnostic DNA sequence and fragment analysis. For clinical diagnostic DNA analysis, it is desirable to examine hundreds of complex DNA samples per day. Existing technology does not provide for such capacity. For example, operation of a typical automated DNA sequencer to evaluate at most about 10 samples requires that a skilled technician spend up to four hours constructing a gel holder, filling the gel holder with actively polymerizing acrylamide solution, inserting a well-forming comb before substantial polymerization has occurred, and waiting for the gel to polymerize (see Maniatis, 1987). Using the gel is equally time consuming. Existing technologies use low density electric fields (less than 100 volts/cm) requiring sample running times of up to four hours. It would be advantageous to have an improved apparatus with improved sample throughput for real-time DNA analysis particularly for use in clinical diagnostic applications.
It is an object of this invention to provide a gel electrophoresis and fluorescence detection apparatus suitable for high throughput clinical diagnostic DNA and protein analysis.
It is a further object of the present invention to provide an electrophoresis apparatus which is capable of using gels which use a high magnitude electric field of 100-400 v/cm.
It is a further object of the invention to provide improved optical excitation and detection techniques for real time detection of fluorescently labeled DNA or protein samples in an electrophoresis gel.